Storage-Induced Micro-Erythrocytes Can Be Quantified and Sorted by Flow Cytometry.

Mickaël Marin,Pascal Amireault,Sandy Peltier,Sonia Georgeault,Pierre A Buffet,Olivier Hermine,Philippe Roingeard,Camille Roussel,Youcef Hadjou,Michaël Dussiot

doi:10.3389/fphys.2022.838138

Abstract

Refrigerated storage of red cell concentrates before transfusion is associated with progressive alterations of red blood cells (RBC). Small RBC (type III echinocytes, sphero-echinocytes, and spherocytes) defined as storage-induced micro-erythrocytes (SME) appear during pretransfusion storage. SME accumulate with variable intensity from donor to donor, are cleared rapidly after transfusion, and their proportion correlates with transfusion recovery. They can be rapidly and objectively quantified using imaging flow cytometry (IFC). Quantifying SME using flow cytometry would further facilitate a physiologically relevant quality control of red cell concentrates. RBC stored in blood bank conditions were stained with a carboxyfluorescein succinimidyl ester (CFSE) dye and incubated at 37°C. CFSE intensity was assessed by flow cytometry and RBC morphology evaluated by IFC. We observed the accumulation of a CFSEhigh RBC subpopulation by flow cytometry that accounted for 3.3 and 47.2% at day 3 and 42 of storage, respectively. IFC brightfield images showed that this CFSEhigh subpopulation mostly contains SME while the CFSElow subpopulation mostly contains type I and II echinocytes and discocytes. Similar numbers of SME were quantified by IFC (based on projected surface area) and by flow cytometry (based on CFSE intensity). IFC and scanning electron microscopy showed that ≥95% pure subpopulations of CFSEhigh and CFSElow RBC were obtained by flow cytometry-based sorting. SME can now be quantified using a common fluorescent dye and a standard flow cytometer. The staining protocol enables specific sorting of SME, a useful tool to further characterize this RBC subpopulation targeted for premature clearance after transfusion.

Highlights

During hypothermic pre-transfusion storage, part of the red blood cells (RBC) metabolism shifts from glycolysis to the pentose phosphate pathway after 10–14 days of storage, leading to a progressive decrease of the intracellular ATP level (D’Alessandro et al, 2015; Bordbar et al, 2016)
This study aimed to develop an alternative method of storage-induced micro-erythrocytes (SME) quantification and sorting using standard flow cytometry
These results show that the CFDA-SE staining protocol with an overnight incubation identifies a discrete RBC subpopulation present in longstored RBC

Summary

Introduction

During hypothermic pre-transfusion storage, part of the RBC metabolism shifts from glycolysis to the pentose phosphate pathway after 10–14 days of storage, leading to a progressive decrease of the intracellular ATP level (D’Alessandro et al, 2015; Bordbar et al, 2016). The fragilized RBC is less capable of coping with the oxidative stress generated by storage, further affecting RBC integrity (Reisz et al, 2016). These metabolic and oxidative stresses contribute to the progressive modifications of RBC properties (Yoshida et al, 2019). The pioneer work of Bessis (1972, 1974) using scanning electron microscopy defined RBC morphological categories. This technique is still used to assess RBC morphology during storage (Berezina et al, 2002; Zehnder et al, 2008; Antonelou et al, 2012; Blasi et al, 2012; D’Alessandro et al, 2012; Roussel et al, 2021)

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